BoneBone MatrixMatrix BiochemistryBiochemistry UCLUCL--20072007

Chris Sharp Charles Salt Centre RJ & AH Orthopaedic Hospital Oswestry PARTPART 11 BoneBone BioBio--MarkersMarkers && TheThe MineralMineral Phase,Phase, MineralisationMineralisation

PART 2 The Organic Phase FibrillarFibrillar && MatricellularMatricellular ProteinsProteins ofof BoneBone Basic Terminology

• amino acids, peptides & • enzymes, isoenzymes & isoforms • propepetides & telopeptides • knock–out “KO” animals , Muscle & Fat

skeletonskeleton comprisescomprises ~~ 12%12% ofof bodybody massmass Composition + Structure

Organic 25% 75% Mineral

Structure Quality PARTPART 11

BoneBone BioBio--MarkersMarkers && TheThe MineralMineral Phase,Phase, MineralisationMineralisation Bone Quality: Bone Mass & Mineral Density

Dual-energy x-ray densitometry DXA The Ideal BONE Marker

• tissue specificity • molecular specificity • reflect a dynamic physiological process ¾ bone formation ¾ bone resorption • clinically meaningful • easy to measure • cheap BioBio--markersmarkers ofof bonebone matrixmatrix turnoverturnover Proteins made by OSTEOBLASTS: – bone alkaline phosphatase isoforms- BALP –-BGP – intact procollagen propeptides- PINP & PICP

Products of OSTEOCLAST activity: – bone acid phosphatase isoforms- BAcP – enzymes used in matrix degradation- cathepsin K – products of breakdown- CTx, ICTP & NTx – collagen crosslinks- Pyr & DPD What can influence a bone marker result ? • Pre-analytical variation ¾¾ analyteanalyte stabilitystability ¾¾ interinter && intraintra--individualindividual variationvariation ¾¾ age,age, gendergender && hormonehormone statusstatus ¾¾ fitness,fitness, exerciseexercise && dietdiet ¾¾ timetime ofof day,day, timetime ofof yearyear (season)(season) ¾¾ bloodblood oror urineurine collectionscollections

• Analytical variation ¾¾ interinter && intraintra--assayassay variationvariation (quality(quality ofof thethe assay)assay) ¾¾ pipettingpipetting skillsskills (expertise(expertise ofof thethe technician)technician) Composite Material

Mineral Phase Organic Phase hydroxyapatite Impacts on matrix Impacts on mineral properties: properties: • matrix stability • nucleation •growth • maturity Bio-mineralisation & Teeth • calcium phosphate

hydroxyapatite or “carbonated apatite”

(Ca,Sr,Mg,Na,H2O,[*])10 (PO4,HPO4,CO3P2O7)6(OH,F,Cl,H2O,O,[*])2 where [*] represents a lattice defect

Ca10(PO4)6(OH)2 Calcium Phosphate “Bony” Structures Conodonts to Bony Fishes

Palaeozoic Era 543-248 Myrs Devonian Period 415-360 Myrs BSE-SEM showing “bone mineral density”

From Prof Alan Boyde Bone mineral density distribution (BMDD)

BMDD in 55 normals

OP post-Alendronate

Normal (23wt%)

Osteomalacia Bone mineral density distribution (BMDD)

Optimal Ca2+ distribution in bone mineral with respect to material quality & bone strength

Ruffoni et al Bone 2007;40:1308 Bone Mineralisation: A balance of Phosphatases & Pyro-phosphates

• Alkaline phosphatase (TNAP) • Nucleoside triphosphate pyrophosphohydrolase (NPP1) • PHOSPHO-1 (phosphatase orphan-1)

PPi P + P Alkaline Phosphatase Overview

• ecto-enzymes • dimeric • in vitro alkaline pH optimum ~ pH 10 • phosphotransferase, dephosphorylates substrates • widespread tissue distribution • bone isoforms involved in bone mineralisation • most commonly requested analyte in clinical chemistry – bio-marker !! Structure of human placental ALP Le Du et al J Biol Chem 2001 Alkaline Phosphatases isoenzymes and isoforms • Four loci = 4 ALP isoenzymes

Tissue Non-specific Intestinal Placental Germ Cell

Bone ~ 4 isoforms Liver ~ 3 isoforms Kidney ~ ? IEF BALP Alkaline Phosphatases Anchoring of BALP into the cell membrane

* Differences between BALP isoforms are due to different glycosylation patterns

* Glycosyl-phosphatidylinositol (GPI) anchor

* GPI-Phospholipase C and D releases ALP from cell Chromatographic serum profiles

A Healthy adult, 174 U/L B Prostate cancer with skeletal metastases, 354 U/L Magnusson et al. Clin Chem 1998 Origin of BALP isoforms in human cortical and cancellous bone

BALP isoforms isolated from SaOS-2 cells

B/I B1 B2 B1x mix

Sharp et al. CCA 2007 & Magnusson et al. JBMR 1999 Alkaline Phosphatases Functions of Bone Alk Phos (BALP)

• Phosphatase activity

– provides Pi for mineralization

– removes pyrophosphate or other inhibitors of mineralization Alkaline Phosphatases Proposed actions in bone

TNAP PPi Pi hydroxyapatite

P N A P N P T 1 Ca2+ NTPs other other ALP removes inhibitors sources sources of mineralisation of PPi of Pi Hypophosphatasia reduced alkaline phosphatase activity • heritable, rare (about 1/100,000) • low serum ALP activity (hypophosphatasemia) • high serum/urine concentrations of PPi • variable severity of skeletal symptoms • poor skeletal calcification, rachitic deformities, fractures, early tooth loss Hypophosphatasia – mutations in TNALP gene that impact on enzyme function Clinical forms: 1 Perinatal – die in utero or shortly after birth 2 Infantile - <6mths, rickets, failure to thrive 3 Childhood – premature loss of teeth 4 Adult – recurrent, poorly healing fractures 5 Odonto HPP – loss of deciduous teeth <3yrs, dental but not skeletal problems

Diagnosis: • plasma pyridoxal phosphate increased • plasma ALP decreased No established treatment Micro-CT images of upper tibae from WT & TNALP-/- mice

Anderson et al. Am J Pathol 2004; 164:841 BoneBone BioBio--markersmarkers 1:1: BALPBALP Bone Specific ALP isoforms – from OSTEOBLASTS Reflects bone formation – elevated in high bone turnover states Easily measured – RIA, ELISA or enzyme activity BALP isoforms in Paget’s Disease of Bone

Coutris Index vs BALP-B1 & B2 Activities 1800

1600

y

t 1400 i 0.064x v

i ♦ B1 y = 13.604e t 1200 c R2 = 0.6 a

1) 000 m L 0.0737x / y = 44.168e or B2

(U 800 □ 2 of R = 0.508 s

i 600

LP 400 A B 200

0 0.0 10.0 20.0 30.0 40.0 50.0 99 Tc -MDP scan % skeletal involvement (Coutris Index) PART 2 The Organic Phase

•• fibrillarfibrillar collagenscollagens -- structuralstructural •• matricellularmatricellular proteinsproteins -- biologicalbiological modulatorsmodulators Gla–Proteins : 1 Bone gla- (osteocalcin) & Matrix Gla-protein

HOOC GLUtamate CH CH2 + COOH ~HN-CH-CO~

HOOC COOH CH

CH2 GLA ~HN-CH-CO~ • -dependent γ- • characteristic Gla-domains Gla–Proteins : 3 BGP or Osteocalcin

Summary • conserved across spieces • specific to osteoblasts & bones/teeth • contains up to 3 Gla sites • binds metal ions, Ca2+, Mg2+ etc. and bone mineral • homology with blood clotting factors • various forms in serum can reflect bone formation and resorption Osteocalcin

C Asp-Glu Helix N

35

45

30 40

10

15 20 25

------Gla Helix Gla – Proteins : 2 Osteocalcin

Hoang QQ et al. Nature 2003; 425:977 structure

interaction with bone mineral

Hoang QQ et al. Nature 2003; 425:977 Gla - Proteins : 5 Function 2 OC KO Mice Lee et al. Cell 2007, 130:456-469 insulin secretion β-cell proliferation become insulin resistant visceral fat

“Energy Regulation”

?? FUNCTION : – bone-derived hormone involved in regulation of energy metabolism Gla - Proteins : 6 Function 1 OC KO Mice Ducy et al. Nature 1996; 238;448 • deletion of OG1 and OG2 from mOC locus • serum OC wt 362, -/- 0 ng/ml • KO (-/-) normal at birth - by 6 months, cancellous & cortical BFR, cortical thickness & density visceral fat

?? FUNCTION : negative regulator of bone formation – inhibits bone formation - analogous to MYOSTATIN Gla - Proteins : 7 ComparisonComparison ofof GlaGla proteinsproteins inin bonebone

Osteocalcin (BGP) Matrix Gla-protein (MGP)

• human gene Chrom 1 • human gene Chrom 12 • bone (5.7kD) • bone & (10.6kD) • 49 aa, fully processed • 84 aa, retains Nt-propep (1-49h bone) (1-77h bone) • 3 Gla sites • 5 Gla sites • binds mineral • binds mineral & matrix • regulates growth (?) • inhibits calcification Gla - Proteins : 8 MGP KO Mice Lou et al. Nature 1997; 386;78

• smaller

• soft tissue calcification

Arterial network FUNCTION : inhibits calcification BoneBone BioBio--markersmarkers 2:2: OsteocalcinOsteocalcin Bone Specific – from OSTEOBLASTS Easily measured – RIA, ELISA Reflects bone formation – can be elevated in high bone turnover states But: fragments & epitopes intact blood resorption? Osteopontin & Bone A bridge between bone cells and bone matrix

• osteoblasts • 44kD, 314 residues • present in bone matrix and other cell types

• binds integrin αvβ3 • dephosphorylated form does not bind O’clasts • OPN inhibits mineralisation

• 30% protein mass of body • maintain structure of tissues • cell adhesion • wound healing • pathology Collagens of the ECM : 1

• 27 different types • 42 genetically distinct α-chains • structuralstructural andand functionalfunctional diversitydiversity •• >1,300>1,300 mutationsmutations inin 2323 // 4242 hColhCol genesgenes

Collagens of the ECM : 3 Fibrillar Collagens - chain compositon

Type I α1[α1(I)3] two forms α2[α1(I)]2 α2(I)

Type II α1[α1(II)]3 two forms

Type III α1 [α1(III)]3 Structure of Type I procollagen & collagen Collagens of the ECM : 5 Post-translational modifications Intracellular events • chain association and helix formation

• hydroxylation - Proline - helix stability Lysine - cross-linking

• glycosylation Collagens of the ECM : 6 Post-translational modifications Extracellular events • propeptide cleavage • - forms LysALD - cross-links • monomer assembly • fibrillogenesis • stabilisation Proteoglycans :1 ~30~30 extraextra--// periperi--cellularcellular PG’sPG’s • tissue organisers • tissue growth & maturation FunctionsFunctions • protein-protein interactions • connective tissue assembly • bind growth factors Proteoglycans :2 SmallSmall LeucineLeucine RichRich ProteinsProteins -- SLRPsSLRPs familyfamily Proteoglycans :3 & Biglycan CS/DS-GAG

N-linked oligos

Core protein

~10 LRRs Cys-loop Cys-loop Proteoglycans :4 ClassClass II -- SLRPsSLRPs Decorin (PG-II) Biglycan (PG-I) • 40 / 130 kD • 40 / 270 kD • CS chains in bone • CS chains in bone • wide distribution, • pericellular localised with Col I • preOB , OB ,Ocytes • osteocytes • KO = thin skin • KO = osteopenia • bind collagen Proteoglycans :5 Proposed interaction of Decorin with collagen trimer • Decorin interacts with the C-terminal region (α1(I)CB6 peptide). • Some Lys/Hyl residues may be essential for binding.

Weber et al. JBC 1996; 271:31767 Proteoglycans : 7 Both Decorin KO and Biglycan KO mice have abnormal collagen fibres in ECM decorin KO biglycan KO mice mice

• lateral fusion • irregular cross- sectional profiles

• fragile skin wild type • reduced bone 90nm content Iozzo, RV. Ann Rev Biochem 1998; 67:609 Collagen Matrix Assembly Transport of collagen trimers and initiation of fibrilogenesis in the vicinity of the cell

fibronectin

I small PG’s

I I I I integrins

I

I

I α11β1

I I α2β1

cell fibronectin scaffold & integrin nucleation centre Velling et al JBC 2002;277:37377 Collagen Crosslinking

helix Pyridinium crosslink

telo

telo Collagen Stabilisation

S T • non-crosslinked A ⇓ B ⇓ I • divalent L ⇓ I T • trivalent Y Collagen Matrix Stabilisation • dependent on 2 enzymes that act on lysine residues:-

• - 3 isoenzymes • lysyl oxidase - 5 isoenzymes Collagen Matrix Stabilisation • lysyl hydroxylase - helix & telopeptide acting isoforms - tissue specific

EC 1.14.11.4 -NH-CH-C=O COOH -NH-CH-C=O COOH CH CO CH CH 2 Asc / Fe++ 2 2 CH2 +CH2 CH2 + CH2

CH2 CH2 O2 HCOH COOH

H2CNH2 COOH CO2 H2CNH2 Lysine αKG HyLys Succ Collagen Matrix Stabilisation • lysyl oxidase -Cu2+ - dependent amine oxidase - forms Lys/HylALD in ¼-stagger collagen

EC 1.4.3.13

-NH-CH-C=O Cu++ -NH-CH-C=O

[CH2]4 +O2 [CH2]3 + NH3 + H2O -NH3+ Pydx Phos HC=O

Lysine / HyLys Lysylald / HyLysald Collagen cross-linking Procollagen HylALD Helical–Lys Hydrox LysALD pathway Telopeptide-Lys Hydrox pathway

bonebone skin, tendon telo-Hyl LYSYL telo-Lys OXIDASE ketoketo--iminesimines aldiminesaldimines divalent XL pyridiniumpyridinium && histidinehistidine adductsadducts pyrrolicpyrrolic crosslinkscrosslinks trivalent XL HHLHHL Known Trivalent Collagen Crosslinks

Pyridinoline Pyrrole Crosslinking at the Ct-telopeptide

Review of Ct-telo structures α2telo non-crosslinked α1telo Ct-telo α1telo InterInter--molecularmolecular ((telotelo –– helix)helix) reduced keto-imine Di-valent DHLNL

Ct-telo IntraIntra--molecularmolecular

Trivalent ((telotelo –– telotelo))

Ct-telo ~40% Pyr Collagen Matrix Stabilisation

Overview • condensations of Lys or Hyl

•Nt- and Ct-telopeptides • inter- and intra-molecular • temporal sequence • type and extent of X-linking influenced by lysyl hydroxylase Bailey AJ. Amino Acids 1991;1:293-306 Collagen , mutations & diseases

• COL1A1, COL1A2 OI, EDS VIIA & VIIB

• COL2A1 chondrodysplasias & osteoarthrosis

• COL3A1 EDS IV Collagen knock-outs 1 Human models osteogenesis imperfecta (OI) • mutations cause dysfunctional or reduced amounts of collagen Spectrum of severity lethal > severe > moderate > mild Type II Types III & IV Type I Osteogenesis Imperfecta

Bullough’s Orthopaedic Pathology 3rd Ed. Collagen knock-outs 2 Animal model • osteogenesis imperfecta mouse (OIM) • natural deletion of COLIA2 gene • only homotrimer type I collagen present Defects (?) Collagen knock-outs 3

Characteristics of the OI Mouse • smaller (25% lighter) • generalised osteopenia • decreased bone strength • fractures • progressively deforming Collagen knock-outs 4 Bone matrix of the OI Mouse

• type I collagen homotrimer [α1(I)3] • disorganised collagen matrix • altered mineral crystal size & composition

Important role of α2(I)-collagen chain in maintaining bone quality Bone Matrix Changes in Osteoarthritis: 1 Quality = composition & structure Bone Structure: histomorphometry Fazzalari Group, Truong et al Arth Res & Ther 2006; 8:R188 Bone Matrix Changes in Osteoarthritis: 2 Quality = composition & structure Matrix Composition: gene expression (mRNA)

Normalised to GAPDH • ↑ALP • ↑OCN • ↑OPN

• ↑COL1A1/COL1A2

Fazzalari Group, Truong et al Arth Res & Ther 2006; 8:R188 BiomarkersBiomarkers 3:3: collagencollagen

PINP PICP

(PINP) Amino propeptide Carboxyl propeptide (PICP)

Helical domain

(Nt-telo) Amino telopeptide Carboxyl telopeptide (Ct-telo) BiomarkersBiomarkers 4:4: typetype II collagencollagen turnoverturnover

location marker Type I collagen Type I collagen formation breakdown divalently XL α1(I).α1(I) 3 telopeptides α1(I).α2(I) 3 ICTP 3 trivalently XL α/β CTx-I 3 telopeptides INTP 3 NTx-I 3 helical domain hydroxyproline 3 NS peptides helical peptide 3 NS collagen trivalent 3 NS cross-links divalent 3 NS propeptides intact PINP 3 PINP-Col1 3 3 Summary Glycoproteins – Alkaline phosphatases, BALP isoforms & role in mineralisation Matricellular proteins Gla-proteins – BGP (OCN) & MGP, possible roles in bone & soft tissues Osteopontin – OPN, role in mineralisation Collagen – Type I, structure, assembly & cross-linking Proteoglycans – Decorin & Biglycan, roles in collagen fibril assembly & organisation